Molecular evidence of potential novel spotted fever group rickettsiae, Anaplasma and Ehrlichia species in Amblyomma ticks parasitizing wild snakes.

BACKGROUND: Amblyomma ticks parasitize a wide range of animals in tropical regions. This study describes the identification of Amblyomma ticks from wild snakes in Malaysia and the detection of potential human pathogens such as Rickettsia, Anaplasma, Ehrlichia and bartonellae in the ticks.
FINDINGS: Twenty one adult ticks (twelve A. varanense and nine Amblyomma helvolum ticks) identified from seven Python molurus snakes in Sepang and a pool of six A. helvolum ticks from a Naja sumatrana snake in Johore, Malaysia were investigated in this study. Amplification of the citrate synthase (gltA), 190-kDa surface antigen gene (ompA), 135-kDa surface antigen (ompB) and surface cell antigen (sca4) genes followed by sequence analysis confirmed the presence of two potential novel spotted fever group rickettsiae in the ticks. Candidatus Rickettsia sepangensis from an engorged A. varanense tick demonstrated high sequence similarity to Rickettsia tamurae; while Candidatus Rickettsia johorensis from two samples (individual and pooled) of A. helvolum and two A. varanense ticks were closely related to Rickettsia raoultii. Anaplasma and Ehrlichia DNA were detected from seven and two ticks, respectively. No bartonellae was detected from any of the ticks.
CONCLUSION: The finding in this study suggests that Amblyomma ticks parasitizing wild snakes may serve as reservoir hosts and carriers for rickettsioses, anaplasmosis and ehrlichiosis in this region.

Background

Ticks are the vector for numerous emerging zoonotic diseases which can be severe and life-threatening to humans. In nature, ticks and a wide range of animals may act as reservoirs or amplifiers for human pathogens such as spotted fever group rickettsiae, anaplasma, ehrlichiae and bartonellae. Humans can be accidentally infected with these organisms through tick bites. The ticks belonging to the genus Amblyomma have been implicated as a carrier for several pathogenic rickettsiae including Rickettsia rickettsii, R. aeschlimannii, R. raoultii, and R. tamurae [1], Anaplasma phagocytophilum, Ehrlichia chaffeensis and E. ewingii [2,3]. Additionally, Bartonella DNA has also been detected in A. americanum ticks [4].

Amblyomma ticks parasitize a wide range of animals and are often seen on mammalian hosts, reptiles and amphibians [5,6]. However, information is lacking on tick carriage of emerging human pathogens in the tropical region. In this study, we assessed the occurrence of these microorganisms in Amblyomma ticks parasitizing wild snakes in Malaysia by using molecular approach.

Methods

Twenty-one adult ticks (12 A. varanense and nine A. helvolum) from seven Python molurus snakes from Sepang (2°49′10.862″N, 101°44′1.262″E) and a pool of six A. helvolum ticks from a Spitting cobra (Naja sumatrana) in Johore, Malaysia (1°43′58.321″N, 103°54′5.082″E) collected from August-October 2012 were investigated in this study. The ticks were identified based on the taxonomic keys of Burridge [5] and Kohls [7].

Tick DNA was extracted using QIAamp DNA mini kit (Qiagen, Hilden, Germany) in accordance to the manufacturer’s instruction. Four rickettsial-specific genes were targeted for amplification from the tick samples, i.e., citrate synthase gene (gltA), 190-kDa outer membrane protein gene (ompA), 135-kDa outer membrane protein gene (ompB) and surface cell antigen (sca4) [8-11]. Identification of Anaplasma and Ehrlichia DNA in the samples was performed using a PCR assay targeting 16S rRNA gene of the organisms [12] followed by sequence analysis. For further differentiation of Anaplasma spp., amplification of the full length sequences of 16S rDNA and msp4 genes were performed [13]. A PCR assay targeting citrate synthase (gltA) gene was performed for detection of bartonellae DNA [14]. Cloned PCR2.1-TOPO T/A plasmids (Invitrogen, USA) with amplified gltA fragment from R. honei (strain TT118), ompA and ompB fragments from rickettsial endosymbionts (98% similarity to R. heilongjiangensis and R. raoultii, respectively) of tick samples were used as positive controls. BLAST analysis was performed to search for homologous sequences in the GenBank database. To determine the phylogenetic position of the rickettsiae identified in this study, dendrogram was constructed based on concatenated sequences of gltA (1040–1046 nucleotides) and ompA (407–431 nucleotides) genes using neighbour-joining method of MEGA software [15].

Findings

Table 1 shows the amplification of rickettsial gltA gene from three A. varanense (S5, S4-2 and S7-2) and two A. helvolum tick samples (S6-1, P1). The gltA and ompA sequences from the S5 tick was almost similar (99.0% and 97.7%, respectively) with R. tamurae strain AT-1 from A. testudinarium tick in Japan [16]. However, the ompB gene of the rickettsia was unable to be amplified and no significant similarity was obtained for the amplified sca4 fragment.

Table 1

Molecular detection of rickettsiae, anaplasma and ehrlichia and blast analysis of the sequences derived from tick samples in this study

Tick sample (Species, location)	Rickettsia				Anaplasma 16S rDNA
	gltA	ompA	ompB	sca4
Candidatus Rickettsia sepangensis
S5(A. varanense, Sepang)	R. tamurae strain AT-1 (AF394896) (1033/1043, 99.0%)	R. tamurae strain AT-1 (DQ103259) (417/427, 97.7%)	Unable to be amplified	No significant similarity	A. phagocytophilum (AY551442, 99%, 253/256), A. platys (JX261979, 99% 253/256)
Candidatus Rickettsia johorensis
P1 (pooled A. helvolum, Johore), S4-2 (A. varanense, Sepang), S6-1 (A. helvolum, Sepang)	R. raoultii strain Khabarovsk (DQ365804) (1057/1060, 99.7%)	R. raoultii strain Khabarovsk (DQ365801) (418/429, 97.4%)	R. raoultii strain Khabarovsk (DQ365798) (762/775, 98.3%)	R. raoultii strain Khabarovsk (DQ365808) (795/816, 97.4%)	Not amplified
S7-2 (A. varanense, Sepang)	R. raoultii strain Khabarovsk (DQ365804) (1057/1060, 99.7%)	R. raoultii strain Khabarovsk (DQ365801) (418/429, 97.4%)	R. raoultii strain Khabarovsk (DQ365798) (762/775, 98.3%)	R. raoultii strain Khabarovsk (DQ365808) (795/816, 97.4%)	A. bovis (AB983438, 99%, 253/256)
S2, S4 (A. helvolum, Sepang), S6, S7 (A. varanense, Sepang)	Not amplified				A. phagocytophilum (AY551442, 99%, 253/256), A. platys (JX261979, 99%, 253/256)
S6-2 (A. varanense, Sepang)	Not amplified				A. bovis (AB983438, 99%, 253/256)
S3, S7-3 (A. varanense, Sepang)	Not amplified				Ehrlichia spp. (J410257, 99%, 249/256)

The sequences obtained for rickettsiae from S5 and P1 ticks have been deposited in the GenBank database under the accession numbers: [gltA (GenBank: KJ769648, KJ769650), ompA (GenBank: KJ769649, KJ769651), ompB (GenBank: KJ769652), sca4 (GenBank: KM977711)].

BLAST analysis of the rickettsial gltA sequence from two samples (individual and pooled) of A. helvolum (S6-1, P1) and two A. varanense (S4-2 and S7-2) ticks demonstrated the closest match (99.7%) to R. raoultii strain Khabarovsk (Table 1), which was cultivated from Dermacentor ticks in Russia and France [17]. The sequence similarity of the ompA, ompB and sca4 sequences of these ticks with those of R. raoultii strain Khabarovsk was 97.4%, 98.3% and 97.4%, respectively.

According to the current criteria for speciation of rickettsial species, uncultured rickettsia exhibiting sequence similarity of ≤99.9% for gltA, ≤ 98.8% for ompA, ≤99.2% ompB and ≤99.3% for sca4 genes with a validated Rickettsia species may be given Candidatus status [18]. Hence, the rickettsiae are thus named as Candidatus Rickettsia sepangensis and Candidatus Rickettsia johorensis, respectively, in accordance to the location of their first sample collection. The dendrogram constructed using concatenated sequence of gltA and ompA gene fragments (Table 2 and Figure 1) confirmed the clustering of Candidatus Rickettsia sepangensis with the type strain of R. tamurae, and Candidatus Rickettsia johorensis with R. raoultii type strains.

Table 2

GenBank accession numbers of the rickettsial gene sequences used for the construction of a concatenated NJ tree

Rickettsia sp.	GenBank accession no. for targeted genes
	gltA	ompA
Rickettsia raoultii strain Elanda-23/95	EU036985	EU036986
Rickettsia raoultii strain Khabarovsk	DQ365804	DQ365801
Rickettsia raoultii strain Marne	DQ365803	DQ365799
Rickettsia aeschlimannii	AY259084	AY259083
Rickettsia massiliae Mtu 1	U59719	U43799
Rickettsia rhipicephali strain HJ5	DQ865206	DQ865208
Rickettsia parkeri	KF782319	KF782320
Rickettsia sibirica 246	U59734	U43807
Rickettsia conorii Seven	U59730	U43806
Rickettsia honei	AF018074	AF018075
Rickettsia rickettsii R (Bitterroot)	U59729	U43804
Rickettsia montana	U74756	U43801
Rickettsia tamurae strain AT-1	AF394896	DQ103259
Rickettsia japonica YM	U59724	U43795
Rickettsia heilongjiangensis strain CH8-1	AB473812	AB473813
Rickettsia felis strain URRWXCal2	AF210692	AF210694
Rickettsia slovaca N.A. 13-B	U59725	U43808
Rickettsia monacensis strain IrR/Munich	DQ100163	DQ100169
Candidatus Rickettsia sepangensis (S5)	KJ769648	KJ769649
Candidatus Rickettsia johorensis (P1)	KJ769650	KJ769651

Figure 1

Phylogenetic placement of concatenated sequences ( and ) of known rickettsial species in Table . Bootstraps analysis was performed with 1000 replications. Numbers in brackets are GenBank accession numbers. Scale bar indicates the nucleotide substitutions per sites.

Several spotted fever group rickettsiae with unknown or potentially pathogenicity for humans have been reported in the Southeast Asia region, mainly in Thailand. R. honei (strain TT-118) and R. thailandii sp. nov. have been identified from Ixodes and Rhipicephalus ticks [19,20]. Closely related species of R. raoultii have also been detected from A. helvolum from a lizard (Varanus salvator) in Thailand [21]. Exposure to infected snake ticks may pose risks to human health as R. tamurae and R. raoultii have been implicated in human infections [22,23]. High antibody prevalence to R. honei (TT118 strain) has been reported in febrile patients in rural areas in Malaysia [24]. However, information on the type of spotted fever group rickettsiae is still lacking.

Anaplasma DNA was amplified from seven ticks (Table 1). Based on the 256 nucleotides of the amplified 16S rDNA partial gene fragments, sequences from three A. varanense and two A. helvolum ticks showed the closest similarity to those of A. phagocytophilum [Genbank accession no.: AY551442, 99%, 253/256] or A. platys [Genbank accession no.: JX261979, 99%, 253/256]. A. bovis DNA [Genbank accession no.:AB983438, 99%, 253/256] was amplified from two A. varanense ticks, whereas DNA of Ehrlichia spp. [Genbank accession no.: KJ410257, 99%, 249/256] was amplified from two A. varanense ticks. Attempts to determine the full length sequence of 16S rRNA and msp4 genes were not successful as the sequences obtained were not satisfactory for analysis. No bartonellae was detected from any of the ticks understudied.

There is no report on the human infections caused by tickborne pathogens with reptile as a host in Southeast Asia. The presence of SFG rickettsiae (Rickettsia species closely related to R. raoultii, R. tamurae and R. bellii) has been recently shown in A. varanense and A. helvolum in Thailand [25]. Detection of R. honei in a reptilian tick, Bothriocroton hydrosauri (formerly Aponomma hydrosauri) has been reported in Australia [26]. Rickettsia spp. closely related to R. tamurae has also been detected in A. fimbriatum ticks collected from reptiles (yellow-spotted monitor, water python and green-tree snake) in the Northern Territory of Australia [27], and A. exornatum tick from a lizard (Varanus olivaceus) in United States of America [28]. In the South America, Rickettsia sp. strain Colombianensi has been identified from A. dissimile ticks parasitizing iguanas in Colombia [29]. All these findings suggest the existing of a natural cycle of spotted fever group rickettsial infection in ticks and snakes in different geographical regions. A. phagocytophilum has been detected in A. flavomaculatum tick collected from a Varanus exanthematicus lizard imported into Poland [30]. Meanwhile, the detection of Ehrlichia spp. from ticks collected from snakes has not been reported previously and thus, merits further investigation.

A. helvolum ticks have been identified from different snakes including Python sp., Ptyas (Zamensis) korros and Naja naja (Kohls) [7] in Malaysia. A. varanense is also one of the most widespread Amblyomma ticks in large snakes in Southeast Asia [5]. As P. molurus and N. sumatrana snakes are native to Southeast Asia [31,32], ticks parasitizing the snakes could be endemic where the animal hosts are available. Although there is no data about the affinity of the ticks to bite humans yet, the detection of rickettsial agents in the snake ticks poses a risk to both wildlife and human. Further work is required to assess the prevalence of these potential tick-borne pathogens on a larger scale.

Conclusions

This study presented the molecular evidence of the presence of potential novel spotted fever group rickettsiae closely related to R. tamurae and R. raoultii, Anaplasma and Ehrlichia spp. in two species of Amblyomma ticks parasitizing P. molurus and N. sumatrana snakes. The finding in this study suggests the potential role of Amblyomma ticks as a reservoir host and carrier for rickettsioses, anaplasmosis and ehrlichiosis in this region.